Electrophysiological and Autoradiographical Evidence of V, Vasopressin Receptors in the Lateral Septum of the Rat Brain

Proc. Nati. Acad. Sci. USA Vol. 84, pp. 7778-7782, November 1987 Neurobiology Electrophysiological and autoradiographical evidence of V, vasopressin receptors in the lateral septum of the rat brain (binding sites/brain slices/oxytocin/receptor subtypes/synthetic structural analogues) M. RAGGENBASS, ELIANE TRIBOLLET, AND J. J. DREIFUSS* Department of Physiology, University Medical Center, 1211 Geneva 4, Switzerland Communicated by Berta Scharrer, July 20, 1987 (received for review May 5, 1987) ABSTRACT Extracellular recordings were obtained from tricular nucleus or from the amygdala (9, 10). Vasopressin single neurons located in the lateral septum, an area known to can be released in a calcium-dependent manner from rat receive a vasopressinergic innervation in the rat brain. Ap- septum in situ (11) and in vitro (12). In addition, binding sites proximately half of the neurons tested responded to 8-L- for vasopressin have been detected by light microscopic arginine vasopressin (AVP) by a marked increase in firing rate autoradiography in the lateral septum (13-16). Taken togeth- at concentrations >1 nM. The effect ofvasopressin was blocked er, these data suggest that vasopressin may function as a by synthetic structural analogues possessing antagonistic prop- peptide transmitter in the lateral septum. erties on peripheral vasopressin and oxytocin receptors. To corroborate this conjecture we have carried out extra- Oxytocin was much less potent than vasopressin in firing septal cellular single-unit recordings from slices of the rat brain and neurons, and a selective oxytocic agonist was totally ineffective. have observed that vasopressin increases the excitability of The action of vasopressin on neuronal firing was mimicked by neurons located in the lateral septum. Next, to characterize the vasopressor agonist [2-phenylalanine,8-ornithinelvasotocin the receptor type(s) mediating this stimulatory effect of but not by the selective antidiuretic agonist 1-deamino[8-D- vasopressin, we have tested various synthetic structural argininelvasopressin. In a parallel study, sites that bind analogues of vasopressin that possess receptor-specific, [31H]AVP at low concentration (1.5 nM) were found by in vitro agonistic properties in peripheral tissues (17). In a parallel autoradiography in the lateral septum. Adjacent sections were study, we determined the distribution of [3H]vasopressin and also incubated with 1.5 mM [3HJAVP and, in addition, with 100 [3H]oxytocin binding sites in the septum by in vitro light nM [2-phenylalanine,8-ornithine]vasotocin or 1-deamino[8-D- microscopic autoradiography and assessed the ligand speci- arginine]vasopressin-i.e., the same compounds as those used ficity of these binding sites by performing displacement tests for the electrophysiological study. Results showed that the using the same structural analogues. vasopressor agonist, but not the antidiuretic agonist, displaced [3H]AVP, thus indicating that the vasopressin binding sites MATERIALS AND METHODS detected by autoradiography in the septum were V1 (vasopressor type) rather than V2 (antidiuretic type) receptors. Based on Electrophysiological Recordings. Experiments were done the electrophysiological evidence, we conclude that these re- using male adult rats (200-300 g) from a Sprague-Dawley- ceptors, when occupied, lead to increased firing oflateral septal derived strain. The animals were decapitated, their brains neurons. were excised, and a block of brain tissue containing the septum was prepared. Coronal slices, 300-400 um thick, The neurohormone vasopressin is synthesized in the hypo- were cut using a vibrating microtome. Slices were transferred thalamus and carried by axoplasmic transport to the to a thermoregulated (34-35°C) recording chamber and neurohypophysis, where the arrival of action potentials placed at the interface between a humidified oxygenated triggers its release from nerve terminals into the bloodstream atmosphere and a perfusion solution of 130 mM NaCI/5 mM (1). Vasopressin then acts upon various peripheral tissues KCI/20 mM NaHCO3/1.2 mM KH2PO4/10 mM glucose/i after its binding to membrane receptors. The hypertensive mM MgSO4/0.7 mM CaCl2. The solution was gassed with effect of vasopressin is mediated by V1 receptors located on 95% 02/5% C02, pH 7.35-7.45. Peptides were tested by vascular smooth muscle; in addition, V1 receptors have been dissolving them in the perfusion solution, which flowed at -2 detected in several other tissues, such as liver, where ml/min and could be exchanged in 2.5-3.0 min. Under vasopressin causes glycogenolysis (2). Binding of vasopres- microscopic guidance, extracellular single-unit recordings sin to V1 receptors stimulates Ca2+ mobilization through a were obtained from the lateral septum using glass micropi- process that involves phosphatidylinositol breakdown (3). pettes filled with 4 M NaCI and having tip dc resistances of The antidiuretic effect of vasopressin is mediated by V2 5-25 MW. Voltage signals were amplified, filtered, and receptors located in the kidney that activate a membrane displayed on an oscilloscope. Records of single-unit activity adenylate cyclase (2). were gathered with a rate meter and monitored on chart. Furthermore, it has become clear that vasopressin is Light Microscopic Autoradiography. The brains of animals synthesized by neurons in hypothalamic and extrahypothal- from the same stock were rapidly removed and frozen in amic nuclei the axons of which project to numerous areas within the mammalian central nervous system (4, 5). In rats, Abbreviations: AVP, 8-L-arginine vasopressin; OXT, oxytocin; [Phe2,Orn']VT, [2-phenylalanine,8-ornithine]vasotocin; HO[Thr4, the lateral division of the septum is densely innervated by Gly7]OXT, [1-(L-2-hydroxy-3-mercaptopropionic acid), 4-threo- vasopressin-immunoreactive axons and axon terminals (6, 7). nine,7-glycine]OXT; deamino-DAVP, 1-deamino[8-D-arginine]vaso- Some of these axons originate in the bed nucleus of the stria pressin; dEt2Tyr(Me)DAVP, [1-(J3-mercapto-43,,B-diethylpropionic terminalis (8), whereas others may derive from the paraven- acid),2-0-methyltyrosine,8-D-arginine]vasopressin; d(CH2)5Tyr- (Me)AVP, [1-(3-mercapto-,8,,3-cyclopentamethylene propionic acid),2-0-methyltyrosine]arginine vasopressin. The publication costs of this article were defrayed in part by page charge *To whom reprint requests should be addressed at: Department of payment. This article must therefore be hereby marked "advertisement" Physiology, University Medical Center, 9, Av. de Champel, in accordance with 18 U.S.C. §1734 solely to indicate this fact. CH-1211 Geneva 4, Switzerland. 7778 Neurobiology: Raggenbass et al. Proc. Natl. Acad. Sci. USA 84 (1987) 7779 Table 1. Endocrine activities (international units per mg) of For V1/V2 receptor discrimination, one series ofsections was AVP, OXT, and agonists incubated with 1.5 nM [3H]AVP; adjacent sections were Vasopressor Antidiuretic incubated with the same concentration ofvasopressin and, in Peptide (V1) (V2) Oxytocic* addition, with 100 nM of either the V1 agonist [2-phenylalanine,8-ornithine]vasotocin ([Phe2,Orn8]VT) or the V2 agonist AVPt 369 323 25.5 1-deamino[8-D-arginine]vasopressin [Phe2,Orn8]VTf 121 1.6 5.3 (deamino-DAVP). Deamino-DAVPt 0.4 1200 2.9 Incubation was followed by two washes in ice-cold incu- OXT§ 4.3 4 486 bation medium and a quick rinse in distilled water. The slides HO[Thr4,Gly7]OXT§ <0.01 0.004 1002 were then dried with cold air, put for 2 hr in a vacuum des- sicator containing paraformaldehyde powder preheated at *Tested on rat uterus in vitro in the presence of 0.5 mM Mg2+. and then an tManning et al. (20). 80°C, placed in x-ray cassette in contact with *Berde et al. (21). tritium-sensitive LKB Ultrofilm for 3-4 mo at 4°C. §Lowbridge et al. (22). Chemicals. AVP and OXT were purchased from Bachem Fine Chemicals (Bubendorf, Switzerland). The synthetic isopentane at -250C. Series of 10- to 15-,um-thick sections structural analogues [Phe2,0M8]VT; deamino-DAVP; HO- were cut, mounted on gelatin-coated slides, dried for 2-12 hr [Thr4,Gly7]OXT; [1-(f-mercapto-f,3B3-diethylpropionic acid),- at 40C, and stored at -80'C until use. Control sections of 2-0-methyltyrosine,8-D-arginine]vasopressin [dEt2Tyr(Me)- kidney were also prepared. Sections were preincubated for 20 DAVP]; and [1-(,3-mercapto-fB3-cyclopentamethylene propi- min in 50 mM Tris HCl (pH 7.4) containing 100 mM NaCl and onicacid),2-0-methyltyrosine]arginine vasopressin [d(CH2)5- 50 nM guanosine 5-triphosphate, then rinsed in 50 mM Tyr(Me)DAVP] were provided by M. M. Manning (Depart- Tris-HCl. Incubation was then carried out for 1 hr in a humid ment of Biochemistry, Medical College of Ohio, Toledo, chamber by covering each section with 100-200 ,ul ofmedium OH). Three of these compounds are agonists possessing of 50 mM Tris-HCl/O.1 mM bacitracin/5 mM MgCI2/0.1% enhanced receptor specificity, as is shown in Table 1. The last bovine serum albumin containing various labeled and unla- two compounds are powerful V1 and oxytocic antagonists beled peptides. and possess extremely weak V2 agonistic activity (18, 19). For differentiation of oxytocin (OXT) and 8-L-arginine For autoradiography, AVP tritiated on the phenylalanyl vasopressin (AVP) binding sites, one series of sections was residue (specific activity, 40 Ci/mmol; 1 Ci = 37 GBq) and incubated with 1.5 nM [3HJAVP, and another series was tritiated OXT labeled on the tyrosyl residue (specific activity, incubated with 3.5 nM [3H]OXT. Adjacent sections were 35 Ci/mmol) were purchased from New England Nuclear. incubated with the same concentration ofone ofthese labeled Their radiochemical purity was checked by HPLC, the peptides and, in addition, with 150 nM of the specific fractions corresponding to the tritiated peptide being sepa- oxytocic agonist [1-(L-2-hydroxy-3-mercaptopropionic ac- rated on an affinity-chromatography column containing neu- id),4-threonine,7-glycine]oxytocin (HO[Thr4,Gly7JOXT).

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